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miRNA diagnosis marker of osteosarcoma

A technique for osteosarcoma and sarcoma, applied in the field of miRNA diagnostic markers

Active Publication Date: 2016-12-07
杨祚璋
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are few studies on the differential expression of miRNAs in osteosarcoma. Finding and identifying miRNAs related to the occurrence of osteosarcoma provides the basis for clinical diagnosis and treatment of miRNAs, and helps the early diagnosis and prognosis evaluation of osteosarcoma to reach a new level.

Method used

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  • miRNA diagnosis marker of osteosarcoma
  • miRNA diagnosis marker of osteosarcoma
  • miRNA diagnosis marker of osteosarcoma

Examples

Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Screening of miRNAs related to osteosarcoma

[0043] 1. Sample acquisition: Collect 10 patients with chondroblastic osteosarcoma and 10 normal people. The subjects were required to fast for at least 12 hours. At room temperature from 7:00 to 8:00 the next morning, 10ml of venous blood was drawn into an ethylenediaminetetraacetic acid (EDTA) anticoagulation tube, and peripheral blood mononuclear cell PBMCs were extracted, and 1ml Trizol was added. Reagents (Invitrogen), mix well, and store specimens at -80°C for RNA extraction. All blood samples and pathological results should be true and reliable, the study was approved by the ethics committee, and the patient informed consent.

[0044] 2. Extraction of RNA from blood monocytes

[0045] Use Invitrogen's RNA extraction kit to extract total RNA. Proceed as follows:

[0046] (1) First add 1ml Trizol to 1×10 7 In the cells, if the frozen cells are directly added to Trizol, there is no need to thaw them. After pipetting...

Embodiment 2

[0062] Example 2 QPCR verification of differentially expressed miRNA-4521

[0063] 1. Select miRNA-4521 for large sample QPCR verification based on the detection result of the miRNA chip. According to the sample collection method in Example 1, 60 samples of normal people and 60 samples of chondroblastic osteosarcoma were selected.

[0064] 2. The RNA extraction process is the same as in Example 1.

[0065] 3. Reverse transcription:

[0066] 1) Mix 10pg-1μg of total RNA template with 2μl 10× buffer, 2μl dATP (10mM), 0.5μl polyA polymerase, 0.5μl ribonuclease (RNase) inhibitor and RNase free water , The final volume is 20μl, and incubate at 37℃ for 1h.

[0067] 2) Add 1μl 0.5μg / μl Oligo(dT) specific RT primer to the reaction tube and incubate at 70℃ for 5min.

[0068] 3) Immediately incubate on ice for at least 2 minutes to disrupt the secondary structure of RNA and primers.

[0069] 4) Combine the above 20μl reaction mixture with 4μl 5× buffer, 1μl dNTP (10mM), 0.5μl M-MLV reverse transc...

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Abstract

The invention discloses an miRNA diagnosis marker of osteosarcoma. The miRNA marker is miRNA-4521. On the basis of high-throughput sequencing, it discovers that the expression of the miRNA-4521 in the blood of a patient with the osteosarcoma is down-regulated; and by conducting large-sample detection by virtue of qPCR, it further proves that the miRNA-4521 is related to the occurrence and development of the osteosarcoma. Therefore, the invention provides a means and a product for achieving the early diagnosis of the osteosarcoma, so that the early interventional therapy of the patient with the osteosarcoma is achieved, and the survival rate and the survival quality of the patient with the osteosarcoma are improved.

Description

Technical field [0001] The present invention belongs to the field of biomedicine and relates to a miRNA diagnostic marker for osteosarcoma, and the specific miRNA diagnostic marker is miRNA-4521. Background technique [0002] Osteosarcoma (Osteosarcoma) is a malignant tumor that originates from the bones, and its incidence ranks first among primary bone malignancies. Osteosarcoma is divided into various compression types. As a subtype of common osteosarcoma, there is no big difference in the clinical treatment and prognosis of other common osteosarcoma. 75% of patients with osteosarcoma are adolescents, more males (male:female 1.5:1), their malignant degree is very high, they are prone to early lung metastasis, and the prognosis is extremely poor. Once discovered, amputations are often used. Even so, patients The 5-year survival rate after surgery is only 5-20%, which brings great mental damage to patients and their families, and also brings a heavy economic burden to society an...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 杨祚璋杨义豪李东奇张娅
Owner 杨祚璋
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